PROJECT SUMMARY This proposal aims to identify alterations in cochlear macrophages and how these cellular changes correlate with the progression of metabolic presbyacusis (strial presbyacusis). Metabolic presbyacusis is a form of age-related hearing loss resulting from degeneration of the stria vascularis. During aging, the strial microvasculature (StvM) has been observed to degenerate, leaving avascular regions. Although StvM loss is closely associated with declines in auditory function, the cellular and molecular mechanisms mediating this pathology are currently unknown. Macrophages have been observed to reside in the stria vascularis, making close contacts with strial vessels and playing a role in regulating their permeability. Our preliminary data shows that macrophages undergo morphological and transcriptomic changes in the stria vascularis with age, suggestive of an activated state that is pro-inflammatory and may contribute to the cochlear pathology of metabolic presbyacusis. Sphingosine-1- phosphate and its receptor (S1P/S1PR) signaling plays a significant role in determination of macrophage activity where high levels of the bioactive signaling molecule, S1P, polarizes macrophages to an anti-inflammatory state. Preliminary gene expression analysis for proteins that regulate S1P synthesis have identified molecular perturbations consistent with a reduction of S1P synthesis in the aged cochlear lateral wall. Our overarching hypothesis is that age-related macrophage dysfunction and associated alterations of S1P/S1PR signaling contribute to strial microvasculature pathology, EP reduction, and auditory function declines. In Aim 1, we will test the hypothesis that age-related macrophage dysfunction is characterized by a shift towards pro- inflammatory activity. Morphological and molecular measures of macrophage activation will be employed to define macrophage dysfunction in the aging stria vascularis. Gene expression analysis of macrophages will be used to define the transcriptomic profile of these activated immune cells with age in the cochlea. In Aim 2, we will test the hypothesis that disruption of S1P/S1PR signaling contributes to macrophage dysregulation in the aged cochlear lateral wall resulting in EP reduction and associated auditory function declines. Macrophage morphology and activation state will be assessed in a mouse model of disrupted S1P/S1PR signaling along with the determination of the effects on overall cochlear lateral wall and auditory function. This study will identify molecular markers of macrophage dysfunction and assess the contribution of altered S1P/S1PR signaling to the pathology of metabolic presbyacusis. Findings from this study will have implications regarding the role of immune cells and S1P/S1PR signaling in metabolic presbyacusis and offer new potential therapeutic targets for treatment. The mentoring team, training plan, and fellowship goals outlined in the proposal will prepare the applicant for a career as an independent investigator.